1. Field of the Invention
The present invention relates to a cell culturing model and a method for drug screening, more particularly, to a cell culturing model and a method using modified liver cells and hepatic stellate cells in high throughput drug screening for hepatic cirrhosis treatment.
2. Description of Related Art
Hepatic cirrhosis is often resulted from the damage to hepatocytes by alcohol and drugs, or virus infection. Damaged hepatocytes release certain signals to activate hepatic stellate cells (HSCs) in liver tissue into myofibroblasts (MFs). MFs are capable of secreting large varieties of extracellular matrix (ECM), including collagen, fibronectin, laminin and proteoglycan. The accumulation of excess extracellular matrix causes liver fibrosis. In the meantime, accumulated extracellular matrix further obstructs hepatocytes from the supply of oxygen and nutrients, and therefore leads to necrosis. To sum up, hepatic cirrhosis is the result of necrosis of hepatocytes and excessive secretion of extracellular matrix by HSCs, due to the imbalance between the rates of degradation and bio-synthesis of extracellular matrix.
Screening methods for anti-hepatic cirrhosis drugs include in vitro screening by cell culture and in vivo screening by animal models. In vitro screening method by cell culture often uses single type of cells, such as HepG2 cell-line, primary rat hepatocytes, or Chang liver cells derived from human liver. Liver-protecting drugs are treated on the alcohol, carbon tetrachloride (CCl4) or α-galactosamine (Galn) damaged cells, and albumin production, MTT analysis, and the activities of glutathione-S-transferase (GST) or cytochrome P450 are measured, which serve as indication of the recovery of damaged cells. Besides, HSCs or MFs can also be used to observe how the drug inhibits their activities. The expression of α-actin and desmin, collagen production, and the activities of MMP-3 (matrix metalloproteease-3) or -9 usually serve as indicators.
Mice, rats, rabbits and canines are commonly used as animal models for liver protecting drug screening. Acute or chronic hepatic cirrhosis can be induced by the treatment of CCl4or Galn, bile duct ligation or Schistosoma japonicum infection. Recovery of liver damaged animals are observed after administering liver-protecting drugs.
However, using animal experiments to demonstrate the efficacy of newly synthesized drugs conflicts he aspects of economy and humanity. Current in vitro cell model uses single type of cells only, either hepatocytes or HSCs, which is not in accordance with the physiological condition of hepatic cirrhosis. Deviation is likely to occur. A selected drug with therapeutic efficacy should be able to enhance the function of hepatocytes, and also inhibit the activation of HSCs and the production of extracelluar matrix. Therefore, it is necessary to develop an appropriate cell model in accordance with the physiological condition of cirrhotic livers.
Present high throughput screening apparatus for new drugs uses single type of monolayer cells. Monolayer-cells cannot display the real physical condition, not to mention performing screening on several types of cells at the same time. As a result, the actual function and the tissue specificity of the drug cannot be accurately detected, and therefore cause the omission of other important uses which will lead to the misconception of effectiveness. This is also the reason for criticism of the high-speed screening technique for new drugs.
The invention is to develop a cell-culturing model for high throughput screening anti-hepatic cirrhosis drugs. The fluorescence released from reporter genes in genetic modified hepatocytes and HSCs can be detected by a fluorescence reader, which provide a quick and sensitive platform for real-time examination of cell condition and therefore increase the accuracy of drug screening. Experiments on animals may also be replaced in the future to significantly decrease the costs and time for developing a new drug screening.